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Amplite® Fluorimetric Sphingomyelinase Assay Kit *Red Fluorescence*

Sphingomyelinase dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Sphingomyelinase Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).
Sphingomyelinase dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Sphingomyelinase Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).
Sphingomyelinase dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Sphingomyelinase Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).
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Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Excitation (nm)
Emission (nm)
Five types of sphingomyelinase (SMase) have been identified based on their cation dependence and pH optima of action. They are lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium-dependent neutral SMase, magnesium-independent neutral SMase, and alkaline SMase. Among the five types, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered major candidates for the production of ceramide in the cellular response to stress. Our Amplite® Fluorimetric Sphingomyelinase Assay Kit provides the most sensitive method for detecting neutral SMase activity or screening its inhibitors. The kit uses Amplite® Red as a fluorogenic probe to indirectly quantify the phosphocholine produced from the hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). It can be used for measuring the SMase activity in blood, cell extracts or other solutions. The fluorescence intensity of Amplite® Red is proportional to the formation of phosphocholine, therefore to the SMase activity. Amplite® Red enables the assay readable either in fluorescence intensity or absorption mode. The kit is an optimized "mix and read" assay that can be used for real time monitoring of Smase activities. Our kit 13622 has been developed for monitoring acid SMase activity.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare Sphingomyelin working solution (50 µL)
  2. Add SMase standards and/or SMase test samples (50 µL)
  3. Incubate at 37°C for 1 - 2 hours
  4. Add Sphingomyelinase working solution (50 µL)
  5. Incubate at RT for 1 - 2 hours
  6. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw one vial (or bottle) of each kit component at room temperature before starting your experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Sphingomyelinase standard solution (10 U/mL):
Add 20 µL of PBS with 0.1% BSA into the vial of Sphingomyelinase Standard (Component F) to make a 10 units/mL Sphingomyelinase standard solution.

2. Amplite™ Red stock solution (200X):
Add 80 µL of DMSO (Component G) into the vial of Amplite™ Red (Component C) to make 200X Amplite™ Red stock solution. Keep from light. Note: The Amplite™ Red is unstable in the presence of thiols (such as DTT and 2-mercaptoethanol). The final concentration of DTT or 2-mercaptoethanol in the reaction should be lower than 10 µM. Amplite™ Red is also unstable at high pH (>8.5). The reactions should be performed at pH 7 - 8. pH 7.4 is recommended for the assay buffer.



Sphingomyelinase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13621

Add 1 µL of 10 units/mL Sphingomyelinase standard solution into 1000 µL Assay Buffer (Component E) to generate a 10 mU/mL Sphingomyelinase standard solution. Take 10 mU/mL Sphingomyelinase standard solution and perform 1:2 serial dilutions to get serially diluted Sphingomyelinase standards (SMase7 - SMase1). Note: Diluted Sphingomyelinase standard solution is unstable. Use within 4 hours.


1. Sphingomyelin working solution:
Add 50 µL of Sphingomyelin (Component B) into 5 mL of SMase Reaction Buffer (Component D) and mix well to make Sphingomyelin working solution. Note: The Sphingomyelin working solution should be used promptly.

2. Sphingomyelinase working solution:
Add 5 mL of Assay Buffer (Component E) into the bottle of Enzyme Mix (Component A) and mix them well. Then, add 25 µL of 200X Amplite™ Red stock solution into the bottle of Enzyme Mix solution to make Sphingomyelinase working solution before starting the assay. Note: The Sphingomyelinase working solution should be used promptly and kept from light; longer storage is likely to cause high assay background.


Table 1. Layout of Sphingomyelinase standards and test samples in a solid black 96-well microplate. SMase = Sphingomyelinase Standards (SMase1 - SMase7, 0.078 to 5 mU/mL), BL = Blank Control, TS = Test Samples. 


Table 2.  Reagent composition for each well.

SMase1 - SMase750 µLSerial Dilutions (0.078 to 5 mU/mL)
BL50 µLAssay Buffer
TS50 µLtest sample
  1. Prepare Sphingomyelinase standards (SMase), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Treat your cells or tissue samples as desired.

  2. Add 50 µL of Sphingomyelin working solution to each well of Sphingomyelinase standard, blank control, and test samples to make the total Sphingomyelin assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Sphingomyelin working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction mixture at 37°C for 1 - 2 hours.

  4. Add 50 µL of Sphingomyelinase working solution to each well of Sphingomyelinase standard, blank control, and test samples to make the total Sphingomyelinase assay volume of 150 µL/well. For a 384-well plate, add 25 µL of Sphingomyelinase working solution into each well instead, for a total volume of 75 µL/well.

  5. Incubate the reaction mixture for 1 - 2 hours at room temperature (protected from light).

  6. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).


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Spectral properties

Excitation (nm)571
Emission (nm)584



View all 6 citations: Citation Explorer
Doxepin mitigates noise induced neuronal damage in primary auditory cortex of mice via suppression of acid sphingomyelinase/ceramide pathway
Authors: Su, Yu-Ting and Meng, Xing-Xing and Zhang, Xi and Guo, Yi-Bin and Zhang, Hai-Jun and Cheng, Yao-Ping and Xie, Xiao-Ping and Chang, Yao-Ming and Bao, Jun-Xiang
Journal: The Anatomical Record (2017)
Riccardin DN induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo
Authors: Li, Lin and Niu, Huanmin and Sun, Bin and Xiao, Yanan and Li, Wei and Yuan, Huiqing and Lou, Hongxiang
Journal: Toxicology and Applied Pharmacology (2016): 175--184
New Aspects of Silibinin Stereoisomers and their 3-O-galloyl Derivatives on Cytotoxicity and Ceramide Metabolism in Hep G2 hepatocarcinoma Cell Line
Authors: Mashhadi Akbar Boojar, Mahdi and Ejtemaei Mehr, Shahram and Hassanipour, Mahsa and Mashhadi Akbar Boojar, Masoud and Dehpour, Ahmad Reza
Journal: Iranian Journal of Pharmaceutical Research (2016): 421--433
Mitochondrial respiration controls lysosomal function during inflammatory T cell responses
Authors: Baixauli, Francesc and Acín-Pérez, Rebeca and Villarroya-Beltrí, Carolina and Mazzeo, Carla and Nunez-Andrade, Norman and Gab, undefined and é-Rodriguez, Enrique and Ledesma, Maria Dolores and Blázquez, Alberto and Martin, Miguel Angel and Falcón-Pérez, Juan Manuel and others, undefined
Journal: Cell metabolism (2015): 485--498
The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism
Authors: Davis, Warren
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2014): 168--179
A high-throughput sphingomyelinase assay using natural substrate
Authors: Xu, Miao and Liu, Ke and Southall, Noel and Marugan, Juan J and Remaley, Alan T and Zheng, Wei
Journal: Analytical and bioanalytical chemistry (2012): 407--414


View all 65 references: Citation Explorer
A novel mitochondrial sphingomyelinase in zebrafish cells
Authors: Yabu T, Shimuzu A, Yamashita M.
Journal: J Biol Chem (2009): 20349
A novel sphingomyelinase-like enzyme in Ixodes scapularis tick saliva drives host CD4 T cells to express IL-4
Authors: Alarcon-Chaidez FJ, Boppana VD, Hagymasi AT, Adler AJ, Wikel SK.
Journal: Parasite Immunol (2009): 210
Alterations of myelin-specific proteins and sphingolipids characterize the brains of acid sphingomyelinase-deficient mice, an animal model of Niemann-Pick disease type A
Authors: Buccinna B, Piccinini M, Prinetti A, Sc and roglio F, Prioni S, Valsecchi M, Votta B, Grifoni S, Lupino E, Ramondetti C, Schuchman EH, Giordana MT, Sonnino S, Rinaudo MT.
Journal: J Neurochem (2009): 105
A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells
Authors: Vasil ML, Stonehouse MJ, Vasil AI, Wadsworth SJ, Goldfine H, Bolcome RE, 3rd, Chan J.
Journal: PLoS Pathog (2009): e1000420
Expression of alkaline sphingomyelinase in yeast cells and anti-inflammatory effects of the expressed enzyme in a rat colitis model
Authors: Andersson D, Kotarsky K, Wu J, Agace W, Duan RD.
Journal: Dig Dis Sci (2009): 1440
Structure and function of sphingomyelinase
Authors: Oda M., undefined
Journal: Yakugaku Zasshi (2009): 1233
ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan
Authors: Hiukka A, Stahlman M, Pettersson C, Levin M, Adiels M, Teneberg S, Leinonen ES, Hulten LM, Wiklund O, Oresic M, Olofsson SO, Taskinen MR, Ekroos K, Boren J.
Journal: Diabetes (2009): 2018
Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471
Authors: Sugimori D., undefined
Journal: J Biosci Bioeng (2009): 293
Neutral sphingomyelinase-induced ceramide accumulation by oxidative stress during carbon tetrachloride intoxication
Authors: Ichi I, Kamikawa C, Nakagawa T, Kobayashi K, Kataoka R, Nagata E, Kitamura Y, Nakazaki C, Matsura T, Kojo S.
Journal: Toxicology (2009): 33
SMase II, a new sphingomyelinase D from Loxosceles laeta venom gland: molecular cloning, expression, function and structural analysis
Authors: de Santi Ferrara GI, Fern and es-Pedrosa Mde F, Junqueira-de-Azevedo Ide L, Goncalves-de-Andrade RM, Portaro FC, Manzoni-de-Almeida D, Murakami MT, Arni RK, van den Berg CW, Ho PL, Tambourgi DV.
Journal: Toxicon (2009): 743